Our results suggested that Mn JAK signaling pathway could act toxic effects on astrocytes. To support the conclusion of Mn cytotoxicity on the cultured astrocytes, the morphology changes were observed by the inverted phase contrast microscope. When compared with the untreated astrocytes, the density of normal cells was decreased obviously. And cell shrinkage, nuclear condensation, and fragmentation were observed gradually after exposure to 125, 250, and 500 M MnCl2. In the study, we further found that MnCl2 inhibited cell proliferation in a concentrationdependent manner. As shown in Fig. 4 and Table 1, cell showed a statistically significant increase in G0/G1 fraction accompanying with a decrease in S phase fraction. These results indicated that Mn could induce a G0/G1 phase cell cycle arrest in astrocytes. MnCl2 decelerated the transition from G1 to S phase, for the number of cells entered S phase decreased. Mn induced G0/G1 phase arrest might be caused by the alteration of cell cyclerelated protein. It had been demonstrated that G0/G1 arrest in A549 cells has the dihydrofolate reductase relationship with the decreasing of Cdk4, Cdk2, and Cyclin A, and the increasing of P53 and Cdks inhibitor WAF1/P21.
Apoptosis is a cell death process that plays a critical opioid receptor role in tissue development, homeostasis, and development of disease. It characterized by unique distinguishing features, including cytoplasmic shrinkage, nuclear fragmentation, DNA cleavage, and plasma membrane blebbing with formation of apoptotic bodies. When compared with control group, the apoptotic cell population increased concentration dependently after Mn treatment. As revealed in the study, apoptosis is the major phenomenon of Mn cytotoxicity upon astrocytes. Riluzole has been shown to slow the progression of symptoms in patients with amyotrophic lateral sclerosis, a neurodegenerative disease which characterized by the progressive loss of motoneurons. Many studies demonstrated that riluzole acted protective effects on neurons. And some data had shown that riluzole can reverse Gluinduced astrocyte injury. However, we are aware that there were few or no reports on the effects of riluzole on astrocyte after Mn exposure. In present study, comparing with the control group, the cytotoxicity did not occur in the GW786034 riluzole control group. It suggested that riluzole acts almost no toxic effects on astrocytes.
When compared with 500 M MnCl2 group, cell viability increased, LDH leakage decreased, and injury of morphology recovered obviously in the riluzole pretreatment group. These results were similar to the results of Dagic et al. They found that riluzole could protect astrocyte to reverse Glu induced toxicity at all times in terms of LDH activity and nitrite levels. It had also demonstrated that it produced a marked increase in the striatalglial fibrillary acidic protein. And manner riluzole may stimulate the production of trophic activities for motoneurons by spinal astrocyte cultures. When compared with the control group, there were no obvious changes on cell cycle and apoptosis in the riluzole control group. This indicated that riluzole could not change the normal cell cycle and apoptosis on astrocytes. After comparing with 500 M MnCl2 group, the percent of G0/G1 phase cell was decreased.